1. Field of the Invention
This invention relates to a filtration method for extracting cell produced antiviral substances (CPAS) from a production broth using cross-flow membrane filtration.
2. Description of the Prior Art
"Cell Produced Antiviral Substances" (CPAS) are defined for purposes of this invention as proteins that inhibit virus multiplication which are produced by (a) infecting animal (including human) cells with animal viruses, (b) exposure of animal cells to mitogens, certain synthetic polynucleotides, etc., or (c) by hybridomas and genetically engineered bacterial cell lines. As used in this invention, CPAS includes interferons of all types, lymphokines, monoclonal antibodies produced by hybridomas, and the products of genetically engineered cell lines. All of these substances are distinguished for the purposes of this invention from viral chemotherapy agents, vaccines, defective interfering particles, viral interference substances, and the like.
Where the CPAS is not produced by hybridomas or genetically engineered cell lines, it must be induced in natural cell lines by a "CPAS Inducer", which is defined for purposes of this invention as any substance which will cause a cell to produce CPAS, including viruses, mitogens, synthetic polynucleotide/RNA sources, tilorone hydrochloride, lymphomycin, purified protein derivative of tuberculin, OK-432, and the like. Animal viruses range in size from 10 to 300 nm and have molecular weights upwards of 1.times.10.sup.6 daltons. They therefore are easily filtered by the cross-flow cells useful in this invention.
CPAS is produced by the cells themselves in a "Production Broth" which is defined for the purposes of this invention as a mixture of producing cells which may be a suspension of hybridomas or genetically engineered CPAS producing bacterial cell lines or a suspension of naturally-occurring animal cells with a CPAS inducer, and which may also contain cellular particles, various molecular species of differing molecular weights and configurations, diluents, salts, and various incubation media.
One of the continuing problems in the production of various CPAS is the extraction of a relatively pure CPAS from the production broth. Conventional extraction techniques either cannot efficiently separate the CPAS from other substances in the production broth, cannot separate the CPAS from some contaminents, or destroy the producing bacteria, hybridomas, or cells of the production broth, making a continuous or semi-continuous flow process impossible.
Cross-flow filtration is a relatively new separation technique. Cross-flow filtration cells or similar filtration cells are disclosed in U.S. Pat. Nos. 4,191,182 - Popovich, et al., and 4,212,742 - Solomon, et al., which, however, do not disclose their use in the manner of the present invention.
Additional patents of interest which are not considered to disclose the present invention, include the following:
U.S. Pat. No. 3,926,797 discloses a blood purifying apparatus having a conventional filtration cell and a conventional dialysis cell.
U.S. Pat. No. 4,216,770 - Cullis, et al., discloses a continuous flow treatment for carbamylating red blood cells wherein (1) the red blood cells and plasma are separated, (2) the red blood cells are mixed with an anti-sickle cell agent, (3) the treated cells are separated from waste material, (4) the treated cells are washed, (5) the washed cells are separated from waste material, and (6) recombined with the plasma and returned to the patient. The separations are apparently by gravity without using filtration cells or dialysis cells.
U.S. Pat. No. 4,223,672 - Terman, et al., discloses a system and apparatus for removal of immunological reactants from blood wherein (1) plasma is separated from whole blood by a cell separator (filter) [see column 2, lines 46-55] or by a centrifuge, (2) the plasma is treated with an immunoabsorbent in a variable volume plasma treatment chamber, (3) platelets are removed from the plasma in a platelet separation "bag" 55 [see column 2, lines 31-34 and column 5, lines 20-24], and (4) the separated platelets and other blood components are recombined. The immunoabsorbent is fixed to a carrier vehicle [column 5, lines 53-58] and centrifugation is described extensively [column 6, line 28 et seq.].
U.S. Pat. No. 3,579,441 - Brown discloses blood purification by dual filtration. FIGS. 1A and 2 disclose using both an ultrafilter 40 with blood flow parallel to the filter elements 43 and a hyperfilter 60. The ultrafiltrate under pressure is fed to the hyperfilter, also parallel to the filtering surfaces. The Abstract of the Disclosure has a brief description of the flow pressure.
U.S. Pat. No. 4,209,392 - Wallace discloses a hemodialysis circuit with a single plasma separator (membrane filter) and a sorbents filter in the closed loop.
U.S. Pat. No. 4,243,532 - Tsuda, et al., discloses separating plasma from whole blood using a Kiil-type filtration cell, and an in-line adsorbent filtration apparatus [FIG. 5] for purifying the plasma, after which it is recombined with the other blood components. Centrifugal separation is disclosed as undesirable [column 5, lines 21-28].
U.S. Pat. No. 3,506,126 - Serfass, et al., discloses a closed recirculating hemodialysis system using a single ultrafilter cell for its water/dialysate filtration and selective adsorbers for waste metabolites.
U.S. Pat. No. 3,939,069 - Granger, et al., discloses a conventional dialysis circuit with one dialysis membrane and an in-line charcoal adsorbing filter.
U.S. Pat. No. 4,013,564 - Nose discloses a conventional dialysis circuit [FIG. 9] and apparatus.